Oxabicycloheptanes and oxabicycloheptenes for the treatment of depressive and stress disorders

ABSTRACT

A method of treating a depressive or stress disorder in a subject afflicted therewith comprising administering to the subject an effective amount of a compound having the structure:

This application is a continuation of U.S. patent application Ser. No15/048,389, filed Feb. 19, 2016, now U.S. Pat. No. 9,833,450, whichclaims the benefit of U.S. Provisional Patent Application Nos.62/279,265, filed Jan. 15, 2016; and 62/118,246, filed Feb. 19, 2015,the contents of each of which are hereby incorporated by reference.

Throughout this application various publications are referenced. Thedisclosures of these documents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Unpredicted aversive stimuli trigger rapid emotional and behavioralreactions, and if persistent, may contribute to the emergence ofdepressive-like symptoms in both animals and humans (Knoll and Carlezon,2010). The lateral habenula (LHb) processes aversive stimuli anddisappointment and drives aversive behaviors (Matsumoto and Hikosaka,2007; Stamatakis and Stuber, 2012). The LHb has been implicated inaddiction (Lecca, S. et al. 2014).

Aversive stimuli, and more generally stressors, increase the activity ofLHb neurons projecting to monoaminergic centers including the ventraltegmental area and the raphe nucleus (Hikosaka, 2010; Matsumoto andHikosaka, 2007; Proulx et al., 2014). Indeed in mice exposed tofoot-shocks, the EPSC paired pulse ratio obtained by optogenetic drivenactivation of LHb terminals onto midbrain GABA neurons is rapidlydecreased indicating a higher glutamate release from LHb axons tomidbrain neuronal populations (Stamatakis and Stuber, 2012). Therepetitive exposure to aversive environmental stimuli produces insteadan aberrant and persistent hyperactivity of LHb neurons, which has beenproven instrumental for the emerging of depressive symptoms (Li et al.,2011; Li et al., 2013) (Meye, Valentinova, Lecca et al., 2015).

The underlying mechanisms implicate distinct mechanisms; firstly aCaMKIIβ- and GluA1-dependent strengthening of AMPA-mediated transmission(Li et al., 2013) (Meye, Valentinova, Lecca et al., 2015); and secondlya reduction in the GABA component of GABA-Glutamate co-release typicalof the entopeduncular nucleus-to-LHb synapse (Proulx et al., 2014;Shabel et al., 2014). Altogether, these findings indicate that theinduction and maintenance of depressive behaviors may require complexand temporally distinct synaptic modifications relying on specificinputs and outputs of LHb neurons (Lecca et al., 2014).

Neuronal excitability throughout the central nervous system relies, atleast in part, on the slow GABA-dependent inhibition mediated by GABABreceptors (GABABRs) and G protein-gated inwardly rectifying potassium(GIRK/Kir3) channels (Luscher et al., 1997). Repeated stressful eventslead to the dysregulation of GIRK-dependent signaling (Lemos et al.,2012a; Lemos at al., 2012b). Furthermore, analysis of GIRK geneexpression and function in rodents (Cornelisse et al., 2007; Lujan etal., 2014), and polymoprhisms in the GIRK gene in human cohorts providestrong arguments for GABAB-GIRK implications in the development ofdepressive symptoms (Bagdy et al., 2012).

SUMMARY OF THE INVENTION

The present invention provides a method of treating a depressive orstress disorder in a subject afflicted therewith comprisingadministering to the subject an effective amount of a compound havingthe structure:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;

    -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby treat thedepressive or stress disorder in the subject.

The present invention also provides a method of preventing or reducingthe severity of a depressive or stress disorder in a subject following atraumatic event comprising:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;

    -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby prevent orreduce the severity of the depressive or stress disorder in a subjectfollowing a traumatic event.

The present invention further provides a method of treating addiction ina subject afflicted therewith comprising administering to the subject aneffective amount of a compound having the structure:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;

    -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby treat theaddiction in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. Schematic summarizing the timeline of thebehavioral tests. Footshock exposure (FsE) was followed by there-exposure to the context (ReC) 1 day after and to the forced swim test(FST) 7 days later.

FIG. 1B: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. Bar graph and scatter plots report theincrease in the percentage of freezing in the FsE group compare to theircontrol during the 5 m re-exposure to the operant chamber (CTRL vs FsE;n=8 vs 8; 1.25±0.82% vs 55.63±8.31%, t-test: t14-6.5; ***p<0.001)

FIG. 1C: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. The FsE mice resulted impaired performingthe FST 7 days after the procedure. The bar graph and scatter plotsrespectively report the reduction in the latency at the first episode ofimmobilization (left,) as well as the increased in the total time spentimmobile (right, CTRL vs FsE; t-test, **p<0.01)

FIG. 1D: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. Schematic representing a sagittal sectionof the mouse brain containing the lateral habenula. The dashed linedelimitated the area of recordings.

FIG. 1E: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. FsE increases the spontaneous firingactivity in LHb neurons recorded in a cell-attached configuration. Therepresentative traces from two different cells in a CTRL and FsE micerespectively show the higher firing activity in the last condition. Thebar graph and plot report the significant increase in the tonic activityin the FsE LHb neuronal population (CTRL vs FsE; n=42 vs 43; 8 vs 8mice; 3.12±0.92 Hz vs 5.84±1.27 Hz; Mann Whitney test; **p=0.007).

FIG. 1F: FsE-induced depressive-like phenotype and neuronalhyperexcitability in the LHb. Current-clamp sample traces (50 pAinjection) and action potentials evoked by current steps (graph on theright), showing the higher excitability recorded in the LHb neurons fromFsE animals (CTRL vs FsE; n=12 vs 14; 3 vs 3 mice; two way ANOVA RM F1,288=4.78, *p=0.03).

FIG. 2A: FsE does not alter quantal excitatory and inhibitory releasebut reduces GABAB-GIRK signaling in the LHb. Sample traces, bar graphsand scatters plot showing a similar mEPSCs activity in LHb neurons fromCTRL and FsE mice (CTRL vs FsE; n=21 vs 20; 5 vs 5 mice; Frequency,4.7±0.8 vs 4.5±0.8, Mann Whitney, p=0.7; Amplitude, 25.7±1.8 vs22.7±1.7, t-test, t39=1.21, p=0.23).

FIG. 2B: FsE does not alter quantal excitatory and inhibitory releasebut reduces GABAB-GIRK signaling in the LHb. No difference betweengroups were detected in the frequency and amplitude of mIPSCs (CTRL vsFsE; n=20 vs 20; 5 vs 5 mice; Frequency, 2.8±0.5 vs 3.6±0.7; t-test,t38=0.86, p=0.4; Amplitude, 44.8±3.7 vs 40.4±3.7; t-test, t38=0.82,p=0.4).

FIG. 2C: FsE does not alter quantal excitatory and inhibitory releasebut reduces GABAB-GIRK signaling in the LHb. Representative traces ofthe holding current record in a voltage clamp configuration (Vc=−50 mV)in CTRL and FsE condition. The I-baclofen in the CTRL sample was around100 pA. Baclofen application (100 μM) in a slice from FsE mouse recorded1 h after the procedure, showed a reduced response. This attenuation inthe peak current resulted completely comparable to CTRL condition 30days after the exposure (white traces). In all examples I-baclofen wasfully reversed by the GABAB antagonist CGP54626. (10 μM). The bar graphand scatter plot reports the long lasting attenuation of GABAB-GIRK R₅currents in the LHb of FsE mice. This plasticity persist for 30 days(CTRL vs FsE 1 hour, 1 day, 7 days, 14 days, 30 days, n=15 vs 12, 12,11, 8, 7; 5 vs 4, 4, 4, 3, 2; 104.5±11.3 vs 55.4±7.2, 59.8±9.6,40.1±9.0, 39.6±6.6, 133.0±26.1; one way ANOVA and Dunnet test,F5,64=9.1, ***p<0.0001, **p<0.001,*p<0.05).

FIG. 2D: FsE does not alter quantal excitatory and inhibitory releasebut reduces GABAB-GIRK signaling in the LHb. Bar graph showing theabsence of difference in the I-baclofen between CTRL and FsE when therecordings were performed in presence of the GIRK blockers barium in thebath (1 mM). The residual current evoked by baclofen, independent byGIRKs is not affected by FsE (CTRL vs FsE, n=5 vs 7, 2 vs 3 mice;21.3±3.7 vs 21.3±9.1; t test, t10=0.002, p=0.9).

FIG. 3A: Aversive experience of different nature trigger plasticity ofGABABGIRK in the LHb. The odor predator exposure paradigm. Sample tracesreports the reduction of baclofen-evoked outward current in LHb neuronsrecorded 1 h after the exposure to fox urine (rightmost trace). The bargraph and scatter plot on the right report the significant reduction ofI baclofen compare to the control situation (CTRL vs OPE, n=13 vs 14, 4vs 4 mice; 110.5±29.1 vs 47.4±7.9; t-test, t25=2.16, *p=0.04).

FIG. 3B: Aversive experience of different nature trigger plasticity ofGABABGIRK in the LHb. The restraint stress procedure. The animal wasconstrained in a plexiglass tube for 1 h. The rightmost trace show arepresentative recording in a RS mouse 1 h after the procedure. The bargraph and the plots on the right show the significant I-baclofenreduction in RS mice compare to CTRL (CTRL vs FsE, n=11 vs 14, 3 vs 4mice; 121.7±14.8 vs 68.3±15.3; t-test, t23-2.46, *p=0.02).

FIG. 4A: FsE depresses GABAB-GIRK signal via their internalization inthe LHb. Schematic summarizing the timeline of the electromicroscopyexperiment.

FIG. 4B: FsE depresses GABAB-GIRK signal via their internalization inthe LHb. Sample tissue in CTRL (top) and FsE (bottom) for GABAB1 (left)and GIRK2 (right).

FIG. 4C: FsE depresses GABAB-GIRK signal via their internalization inthe LHb. Bar graphs showing that FsE decreases the distribution ofGABAB1 at the surface (left), increasing its availability at theintracellular compartment (middle) without affects the total amount(right).

FIG. 4D: FsE depresses GABAB-GIRK signal via their internalization inthe LHb. GIRK2 Rs are strongly internalized after FsE. Similarly to theGABAB1 the total amount of GIRK2 Rs is not changed.

FIG. 4E: FsE depresses GABAB-GIRK signal via their internalization inthe LHb. Sample traces and bar graph with plots show the G-protein β andγ subunits activation through the GTPγS in the internal solution (100μM) evoking a Ba²⁺-sensitive outward current (1 mM). In line with EMresults, independently by GABAB activation, the currents induced byGTPγS resulted reduced in FsE mice (CTRL vs FsE; n=6 vs 6; 3 vs 3 mice;92.55±21.83 pA vs 38.00±5.27 pA; t-test, t10=2.43, *p=0.03).

FIG. 5A: PP2A inhibition normalizes GABAB-GIRK function in the LHb afterFsE. Sample traces recorded in LHb neurons from CTRL and FsE in presenceof the PP2A/PP1 inhibitor Okadaic Acid (100 nM) in the internalsolution. Bar graph and scatter plot reporting no difference betweengroups on the I-baclofen current (CTRL vs FsE; n=8 vs 6, 3 vs 2 mice;130.7±25.25 pA vs 120.4±25.61 pA; test, t12=0.28, p=0.78).

FIG. 5B: PP2A inhibition normalizes GABAB-GIRK function in the LHb afterFsE. CaMKII inhibitor KN93 (10 μM) in the internal solution does notaffect the FsE induced reduction of GABAB-GIRK currents. Representativetraces and bar graph with scatter plot were shown (CTRL vs FsE; n=7 vs8, 2 vs 3 mice; 106.3±18.37 pA vs 57.810.38 pA; t-test, t13=2.37,*p<0.03).

FIG. 5C: PP2A inhibition normalizes GABAB-GIRK function in the LHb afterFsE. Sample traces and graph show that LB100, a selective cell permeablePP2A inhibitor applied in the bath (0.1 μM), is sufficient to rescue theFsE-induced inhibition of GABAB-GIRK currents (CTRL vs FsE; n=9 vs 9; 3vs 3 mice; 88.41±18.64 pA vs 85.09±0.67 pA; ttest, t16=0.15, p=0.88).

FIG. 5D: PP2A inhibition normalizes GABAB-GIRK function in the LHb afterFsE. When applied in the bath, LB100 restored the Ba²⁺ sensitive I-GTPγSin FsE animals. Traces and bar graph with singles plot reporting nodifference between groups (CTRL vs FsE; n=6 vs 6; 3 vs 3 mice;94.27±13.29 pA vs 110.6±20.79 pA; t-test, t10=0.66, p=0.52).

FIG. 6A: FsE-mediated hyperexcitability: loss of GABAB control on firingrate and role of PP2A activity. Example traces showing the inhibitoryeffect of baclofen application on the firing activity of LHb neuronsrecorded in a CTRL mouse (upper black trace). The GABAB antagonistCGP54626 (10 μM) revert the baclofen inhibition. Remarkably thismodulatory effect of GABAB agonist and antagonist on the firing of LHbcell is reduced after the FsE (lower red trace). In the middle, thetimecourse of the same examples is reported as 100% of the baseline. Onthe bottom, the bar graphs and scatter dot plots showing the significantchange in firing rate in presence of baclofen and the further increaseobtain with CGP in CTRL (Baseline vs Baclofen, CGP; n=21 vs 10, 11; 4mice; 1.86±0.40 Hz vs 0.33±0.11 Hz, 3.62±0.65 Hz; one way ANOVA andDunnett's test, F2,41=9.52, *p<0.05). This tuning of the firing activityis not more present in the FsE condition (Baseline vs Baclofen, CGP;n=20 vs 9, 11; 4 mice; 6.69±1.13 Hz vs 4.89±1.40 Hz, 8.23±2.25 Hz; oneway ANOVA and Dunnett's test, F2,39=0.87, p>0.05).

FIG. 6B: FsE-mediated hyperexcitability: loss of GABAB control on firingrate and role of PP2A activity. Current-clamp sample traces (20 pAinjected) showing the different modulation of evoked action potentialsby GABAB activation/inactivation. The graph in the middle illustratesthe timecourse effect of the upper examples revealing a strongattenuation of the inhibitory influence of baclofen in FsE mice. The bargraph and scatter plot on the bottom described this effect in a largerpopulation.

FIG. 6C: FsE-mediated hyperexcitability: loss of GABAB control on firingrate and role of PP2A activity. The hyperexcitability of LHb induced byFsE was completely abolished when LB100 (0.1 μM) was applied in thebath. Sample traces (50 pA injected) and graph on the left show theincrease in excitability in FsE mice when the recordings were performedsimply in ACSF and AMPA, NMDA, GABAA blockers (CTRL vs FsE; n=14 vs 17;4 vs 5 mice; two way ANOVA RM F1,348=7.41, *p=0.02). Adding PP2Aactivity inhibitor (sample traces and graph on the right) was sufficientto abolish FsE induced enhancement of excitability (CTRL vs FsE; n=20 vs16; 5 vs 4 mice; two way ANOVA RM F1,408=7.41, p=0.81).

FIG. 7A: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. Schematic illustrating the in vivo experimental protocol andthe timeline of experiments.

FIG. 7B: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. Sample traces, graph and scatter plot of the I-baclofenrecorded one day after the FsE in animals treated with saline (sal) orLB100 (LB, 1.5 mg/kg ip). The I-baclofen attenuation recorded in FsEmice treated with sal (CTRL vs FsE; n=6 vs 7, 3 vs 3 mice; 96.75±19.64pA vs 47.39±11.82 pA; t-test, t11=2.23, *p=0.04) is not present in theFsE group injected with LB (CTRL vs FsE; n=5 vs 10, 2 vs 3 mice;93.88±18.37 pA vs 97.00±18.05 pA; t-test, t13-0.11, p=0.91).

FIG. 7C: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. The reduction of GABAB-GIRK functionality was still present 7days after the shock exposure in animal treated with sal (CTRL vs FsE;n=8 vs 14, 3 vs 4 mice; 99.85±16.38 pA vs 58.31±7.62 pA; t-test,t20=2.62, *p=0.016). A single injection of LB100 6-8 hours after theshock was able to revert the FsE-induced a decrease in I-baclofen 7 dayslater. (CTRL vs FsE; n=8 vs 9, 3 vs 3 mice; 95.54±30.00 pA vs92.97±14.84 pA; t-test, t15=0.079, p=0.93).

FIG. 7D: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. Sample traces (50 pA injected) and input-output curveobtained one day after the protocol and the treatment, reporting thehigher excitability in FsE mice treated with sal compared to theircontrols (CTRL vs FsE; n=14 vs 13; 3 vs 4 mice; two way ANOVA RMF1,300=4.71, *p=0.04). Animals treated with LB100 did not show anydifference in the input-output curve (CTRL vs FsE; n=15 vs 12; 4 vs 3mice; two way ANOVA RM F1,300=0.43, p=0.52).

FIG. 7E: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. Same as FIG. 7D but the in vitro recordings were performed 7days after. The excitability in animal treated with sal still differsbetween CTRL and FsE (CTRL vs FsE; n=15 vs 24; 3 vs 5 mice; two wayANOVA RM F1,444=5.19, *p=0.02). As at day 1 also at day 7 a singleinjection of LB100 6-8 hours after the FsE was able to abolish theincrease in excitability (CTRL vs FsE; n=16 vs 19; 3 vs 4 mice; two wayANOVA RM F1,396=0.20, p=0.65).

FIG. 7F: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. LB100 ip treatment did not affect the freezing score in there-exposure to the context test.

FIG. 7G: In vivo inhibition of PP2A activity rescues GABAB-GIRKfunction, the hyperexcitability and FsE-driven depressive-likephenotype. Bar graph showing that LB100 ip treatment 6-8 h after the FsEis sufficient to rescue the impairment in the Forced swim test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating a depressive orstress disorder in a subject afflicted therewith comprisingadministering to the subject an effective amount of a compound havingthe structure:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;        -   R₅ and R₆ taken together are ═O;        -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby treat thedepressive or stress disorder in the subject.

In some embodiments, the method wherein the amount of the compound iseffective to reduce a clinical symptom of the depressive or stressdisorder in the subject.

In some embodiments, the method wherein the treating comprises reducingthe activity of the lateral habenula of the subject.

In some embodiments, the method wherein the treating comprises reducingthe activity of neurons in the lateral habenula of the subject.

In some embodiments, the method wherein the treating comprises reducingneuronal hyperexcitability in the lateral habenula of the subject.

In some embodiments, the method wherein the treating comprises restoringnormal GABAB-GIRK function in the lateral habenula of the subject.

In some embodiments, the method wherein the treating comprisesinhibiting phosphatase activity in the lateral habenula of the subject.

The present invention also provides a method of preventing or reducingthe severity of a depressive or stress disorder in a subject following atraumatic event comprising:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;

    -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby prevent orreduce the severity of the depressive or stress disorder in a subjectfollowing a traumatic event.

In some embodiments, the method wherein the amount of the compound iseffective to reduce a clinical symptom of the depressive or stressdisorder in the subject.

In some embodiments, the method wherein the preventing or reducingcomprises reducing the activity of the lateral habenula of the subject.

In some embodiments, the method wherein the preventing or reducingcomprises reducing the activity of neurons in the lateral habenula ofthe subject.

In some embodiments, the method wherein the preventing or reducingcomprises reducing neuronal hyperexcitability in the lateral habenula ofthe subject.

In some embodiments, the method wherein the preventing or reducingcomprises restoring normal GABAB-GIRK function in the lateral habenulaof the subject.

In some embodiments, the method wherein the preventing or reducingcomprises inhibiting phosphatase activity in the lateral habenula of thesubject.

In some embodiments, the method wherein the depressive or stressdisorder is a depressive disorder.

In some embodiments, the method wherein the depressive or stressdisorder is a stress disorder.

In some embodiments, the method wherein the depressive disorder is majordepression, dysthymia, postpartum depression, seasonal affectivedisorder, atypical depression, psychotic depression, bipolar disorder,premenstrual dysphoric disorder, situational depression or adjustmentdisorder with depressed mood.

In some embodiments, the method wherein the stress disorder ispost-traumatic stress disorder (PTSD), acute stress disorder,generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD),panic disorder, social phobia or social anxiety disorder.

In some embodiments, the depressive or stress disorder is caused by anaddictive substance.

In some embodiments, the depressive or stress disorder is caused byalcohol or cocaine.

In some embodiments, the depressive or stress disorder is induced bywithdrawal from an addictive substance.

In some embodiments, the depressive or stress disorder is induced bywithdrawal from alcohol or cocaine.

In some embodiments, the depressive or stress disorder is induced bydiscontinuing use of an addictive substance.

In some embodiments, the depressive or stress disorder is induced bydiscontinuing use of alcohol or cocaine.

In some embodiments, the subject is addicted to an addictive substance.

In some embodiments, the subject is addicted to alcohol or cocaine.

In some embodiments, the subject is suffering from withdrawal from theuse an addictive substance.

In some embodiments, the subject is suffering from withdrawal from theuse of alcohol or cocaine.

In some embodiments, the addictive substance is alcohol or cocaine.

The present invention further provides a method of treating addiction ina subject afflicted therewith comprising administering to the subject aneffective amount of a compound having the structure:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ is OH, O⁻, OR₉, O(CH₂)₁₋₆R₉, SH, S⁻, or SR₉,        -   wherein R₉ is H, alkyl, alkenyl, alkynyl or aryl    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H alkyl, alkenyl, alkynyl,        -   aryl,

-   -   -    —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,            -   wherein each R₁₁ is independently H, alkyl, alkenyl or                alkynyl;

    -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H,

or a salt, zwitterion, or ester thereof, so as to thereby treat theaddiction in the subject.

In some embodiments, the addiction is alcohol addiction, drug addiction,stimulant addiction, or nicotine addiction.

In some embodiments, the addiction is cocaine addiction.

In some embodiments, the method wherein the compound has the structure:

In some embodiments, wherein bond α in the compound is present.

In some embodiments, wherein bond α in the compound is absent.

In some embodiments, wherein

-   -   R₃ is OH, O⁻, or OR₉,        -   wherein R₉ is alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, alkenyl, alkynyl,        -   aryl,

In some embodiments, wherein

-   -   wherein    -   R₃ is OH, O⁻ or OR₉,        -   where R₉ is H, methyl, ethyl or phenyl.

In some embodiments, wherein

-   -   wherein    -   R₃ is OH, O⁻ or OR₉,        -   wherein R₉ is methyl.

In some embodiments, wherein

-   -   wherein    -   R₄ is

In some embodiments, wherein

-   -   wherein R₄ is

-   -   -   wherein R₁₀ is H, alkyl, alkenyl, alkynyl, aryl,        -   or

In some embodiments, wherein

-   -   wherein R₄ is

-   -   -   wherein R₁₀ is —H, —CH₃, —CH₂CH₃,            -   or

In some embodiments, wherein

-   -   wherein R₄ is

In some embodiments, wherein

-   -   wherein R₄ is

-   -   -   wherein R₁₀ is H, alkyl, alkenyl, alkynyl, aryl,

In some embodiments, wherein

-   -   wherein R₄ is

In some embodiments, wherein

-   -   wherein R₄ is

In some embodiments, wherein the compound has the structure

-   -   wherein    -   bond α is present or absent;    -   R₉ is present or absent and when present is H, alkyl, alkenyl,        alkynyl or phenyl; and    -   X is O, NR₁₀, NH⁺R₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, substituted alkyl,            alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,            aryl,

-   -   -   —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂,            -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments, wherein the compound has the structure

-   -   wherein    -   bond α is present or absent;

X is O or NR₁₀,

where each R₁₀ is independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl,

-   -   —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂,        -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments, wherein the compound has the structure

-   -   wherein    -   bond α is present or absent;    -   X is O or NH⁺R₁₀,        -   where R₁₀ is H, alkyl, substituted alkyl, alkenyl,            substituted alkenyl, alkynyl, substituted alkynyl, aryl,

-   -   -   —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂.            -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments, wherein the compound has the structure

or a salt or ester thereof.

In some embodiments, wherein the compound has the structure

or a salt or ester thereof.

In some embodiments, the method wherein the subject has been diagnosedwith the depressive or stress disorder.

In some embodiments, the method wherein the subject has beenaffirmatively diagnosed with the depressive or stress disorder.

In some embodiments, the method wherein the subject has beenaffirmatively diagnosed with the depressive or stress disorder bypsychological evaluation or by meeting the criteria in the Diagnosticand Statistical Manual of Mental Disorders.

In some embodiments, the method wherein the subject is a human.

In some embodiments, the method wherein the amount of the compoundadministered to the subject is 0.1 mg/m² to 5 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is 0.25 mg/m^(z2) to 2.5 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is 2.5 mg/m² to 5 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is 3 mg/m² to 4.5 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is about 0.25 mg/m², 0.5 mg/m², 0.83 mg/m²,1.25 mg/m², 1.75 mg/m² or 2.33 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is about 0.25 mg/m², 0.5 mg/m², 0.75 mg/m²,1.0 mg/m², 1.25 mg/m², 1.5 mg/m², 1.75 mg/m², 2.0 mg/m², 2.25 mg/m², 2.5mg/m² or 2.75 mg/m².

In some embodiments, the method wherein the amount of the compoundadministered to the subject is about 3 mg/m², 3.25 mg/m², 3.5 mg/m²,3.75 mg/m², 4 mg/m², 4.25 mg/m² or 4.5 mg/m².

In some embodiments, the method wherein the amount of the compound isadministered once daily.

In some embodiments, the method wherein the amount of the compound isadministered once daily for at least 1 week.

In some embodiments, the method wherein the amount of the compound isadministered once daily for at least 1 month.

In some embodiments, the method wherein the amount of the compound isadministered once daily for at least 1 year.

In some embodiments, the method wherein the amount of the compound isadministered once daily for a three day period.

In some embodiments, the method wherein the amount of the compound isadministered three times per week.

In some embodiments, the method wherein the amount of the compound isadministered on three separate days during a seven day period.

In some embodiments, the method wherein the amount of the compound isadministered on three separate days during a twenty-one day treatmentcycle.

In some embodiments, the method wherein the amount of the compound isadministered on three separate days during week 1 of a twenty-one daytreatment cycle.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated one or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated one or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated two or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated three or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated four or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated five or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated six or more times.

In some embodiments, the method wherein the amount of the compound isadministered on days 1, 2 and 3 of a twenty-one day treatment cycle andthe cycle is repeated between 1 to 10 times.

In some embodiments, the method wherein the amount of the compound isadministered once weekly.

In some embodiments, the method wherein the amount of the compound isadministered twice weekly.

In some embodiments, the method wherein the amount of the compound isadministered once monthly weekly.

In some embodiments, the method wherein of the compound is administeredonce weekly or twice weekly for 1 to 6 weeks.

In some embodiments, the method wherein the amount of the compound isadministered once monthly for 1 to 6 months.

In some embodiments, the method comprising administering to the subjectan amount of 0.25 mg to 7.5 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 0.25 mg to 4 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 0.25 mg to 3 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 0.25 mg to 2 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 0.25 mg to 1 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 1 mg to 5 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 1 mg to 4 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 1 mg to 3 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 1 mg to 2 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 4 mg to 7.5 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 4 mg to 5 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 5 mg to 6 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 6 mg to 7 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 7 mg to 7.5 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of 4.8 mg to 7.2 mg of the compound.

In some embodiments, the method comprising administering to the subjectan amount of about 0.4 mg, 0.8 mg, 1.3 mg, 2 mg, 2.8 mg or 3.7 mg of thecompound.

In some embodiments, the method comprising administering to the subjectan amount of about 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75mg, 2 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4mg, 4.25 mg, 4.5 mg, 4.75 mg, 5 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6 mg, 6.25mg, 6.5 mg, 6.75 mg, 7 mg, 7.25 mg or 7.5 mg of the compound.

In some embodiments of the method, the compound has the structure:

In some embodiments of the method, bond α in the compound is present.

In some embodiments of the method, bond α in the compound is absent.

In some embodiments of the method, the compound wherein

-   -   R₃ is OH, O⁻, or OR₉,        -   wherein R₉ is alkyl, alkenyl, alkynyl or aryl;    -   R₄ is

-   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,

where each R₁₀ is independently H, alkyl, alkenyl, alkynyl, aryl,

In some embodiments of the method, the compound wherein

-   -   R₃ is OH, O⁻ or OR₉,        -   where R₉ is H, methyl, ethyl or phenyl

In some embodiments of the method, the compound wherein

-   -   R₃ is OH, O⁻ or OR₉,        -   where R₉ is methyl.

In some embodiments of the method, the compound wherein

-   -   R₄ is

In some embodiments of the method, the compound wherein

-   -   R₄ is

-   -   -   wherein R₁₀ is H, alkyl, alkenyl, alkynyl, aryl,            -   or

In some embodiments of the method, the compound wherein

-   -   R₄ is

-   -   -   wherein R₁₀ is —H, —CH₃, —CH₂CH₃,            -   or

In some embodiments of the method, the compound wherein

-   -   R₄ is

In some embodiments of the method, the compound wherein

-   -   R₄ is

-   -   -   wherein R₁₀ is H, alkyl, alkenyl, alkynyl, aryl,

In some embodiments of the method, the compound wherein

-   -   R₄ is

In some embodiments of the method,

-   -   wherein R₄ is

In some embodiments of the method, the compound has the structure:

-   -   wherein    -   bond α is present or absent;    -   R₉ is present or absent and when present is H, alkyl, alkenyl,        alkynyl or phenyl; and    -   X is O, NR₁₀, NH⁺R₁₀ or N⁺R₁₀R₁₀,        -   where each R₁₀ is independently H, alkyl, substituted alkyl,            alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,            aryl,

-   -   -   —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂,            -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments of the method, the compound has the structure:

-   -   wherein    -   bond α is present or absent;

X is O or NR₁₀,

where each R₁₀ is independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl,

—CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂,

-   -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments of the method, the compound has the structure:

-   -   wherein    -   bond α is present or absent;    -   X is O or NH⁺R₁₀,        -   where R₁₀ is H, alkyl, substituted alkyl, alkenyl,            substituted alkenyl, alkynyl, substituted alkynyl, aryl,

-   -   -   —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂,            -   where R₁₂ is H or alkyl,

or a salt, zwitterion or ester thereof.

In some embodiments of the method, the compound has the structure:

or a salt or ester thereof.

In one embodiment, the compound of the method has the structure:

or a salt, zwitterion, or ester thereof.

In one embodiment, the compound of the method has the structure:

or a salt, zwitterion, or ester thereof.

In one embodiment, the compound of the method has the structure:

or a salt, zwitterion, or ester thereof.

In one embodiment, the compound of the method has the structure:

or a salt, zwitterion, or ester thereof.

In one embodiment, the compound of the method has the structure:

-   -   wherein    -   bond α is present or absent;    -   R₁ and R₂ together are ═O;    -   R₃ and R₄ are each different, and each is O(CH₂)₁₋₆R₉ or OR₉,    -   or

-   -   -   where X is O, S, NR₁₀, N⁺HR₁₀ or N⁺R₁₀R₁₀,

where each R₉ is H, alkyl, C₂-C₁₂ alkyl substituted alkyl, alkenyl,alkynyl, aryl, (C₆H₅)(CH₂)₁₋₆(CHNHBOC)CO₂H, (C₆H₅)(CH₂)₁₋₆(CHNH₂) CO₂H,(CH₂)₁₋₆(CHNHBOC)CO₂H, (CH₂)₁₋₆(CHNH₂) CO₂H or (CH₂)₁₋₆CCl₃,

-   -   where each R₁₀ is independently H, alkyl, hydroxyalkyl, C₂-C₁₂        alkyl, alkenyl, C₄-C₁₂ alkenyl, alkynyl, aryl,

-   -   —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁,        -   where each R₁₁ is independently alkyl, alkenyl or alkynyl,            each of which is substituted or unsubstituted, or H;    -   or R₃ and R₄ are each different and each is OH or

-   -   -   R₅ and R₆ taken together are ═O;

    -   R₇ and R₈ are each H; and

    -   each occurrence of alkyl, alkenyl, or alkynyl is branched or        unbranched, unsubstituted or substituted,

or a salt, zwitterion, or ester thereof.

In one embodiment, the compound of the method has the structure:

In one embodiment of the method, the bond α is present.

In one embodiment of the method, the bond α is absent.

In one embodiment of the method,

R₃ is OR₉ or O(CH₂)₁₋₆R₉,

-   -   where R₉ is aryl, substituted ethyl or substituted phenyl,    -   wherein the substituent is in the para position of the phenyl;

R₄ is

-   -   where X is O, S, NR₁₀, or N⁺R₁₀R₁₀.    -   where each R₁₀ is independently H, alkyl, hydroxyalkyl,        substituted C₂-C₁₂ alkyl, alkenyl, substituted C₄-C₁₂ alkenyl,        alkynyl, substituted alkynyl, aryl,

-   -   —CH₂CN, —CH₂CO₂R₁₁, —CH₂COR₁₁,

where R₁₁ is alkyl, alkenyl or alkynyl, each of which is substituted orunsubstituted, or H;

or where R₃ is OH and R₄ is

In one embodiment of the method,

R₄ is

-   -   where R₁₀ is alkyl or hydroxylalkyl.

In one embodiment of the method,

-   -   R₁ and R₂ together are ═O;    -   R₃ is OR₉ or O(CH₂)₁₋₂R₉,

where R₉ is aryl, substituted ethyl, or substituted phenyl, wherein thesubstituent is in the para position of the phenyl;

-   -   R₄ is

-   -   -   where R₁₀ is alkyl or hydroxyl alkyl;

    -   R₅ and R₆ together are ═O; and

    -   R₇ and R₈ are each independently H.

In one embodiment of the method,

-   -   R₁ and R₂ together are ═O;    -   R₃ is O(CH₂)R₉, or OR₉,        -   where R₉ is phenyl or CH₂CCl₃,

-   -   R₄ is

-   -   -   where R₁₀ is CH₃ or CH₃CH₂OH;

    -   R₅ and R₆ together are ═O; and

    -   R₇ and R₈ are each independently H.

In one embodiment of the method,

-   -   R₃ is OR₉,        -   where R₉ is (CH₂)₁₋₆(CHNHBOC) CO₂H, (CH₂)₁₋₆(CHNH₂) CO₂H, or            (CH₂)₁₋₆CCl₃.

In one embodiment of the method,

-   -   R₉ is CH₂(CHNHBOC)CO₂H, CH₂(CHNH₂)CO₂H, or CH₂CCl₃.

In one embodiment of the method,

R₉ is (C₆H₅)(CH₂)₁₋₆(CHNHBOC) CO₂H or (C₆H₅)(CH₂)₁₋₆(CHNH₂)CO₂H.

In one embodiment of the method,

R₉ is (C₆H₅)(CH₂)(CHNHBOC)CO₂H or (C₆H₅)(CH₂)(CHNH₂)CO₂H.

In one embodiment of the method,

-   -   R₃ is O(CH₂)₁₋₆R₉ or O(CH₂)R₉,        -   where R₉ is phenyl.

In one embodiment of the method,

-   -   R₃ is OH and R₄ is

In one embodiment of the method,

-   -   R₄ is

-   -   -   wherein R₁₀ is alkyl or hydroxyalkyl.

In one embodiment of the method, R₁₁ is —CH₂CH₂OH or —CH₃.

In one embodiment of the method, the compound has the structure:

or a salt, zwitterion, or ester thereof.

In one embodiment of the method, the compound has the structure:

or a salt, zwitterion, or ester thereof.

The present invention also provides a package comprising:

-   -   a) a pharmaceutical composition comprising an amount of a        phosphatase inhibitor of the present application and a        pharmaceutically acceptable carrier; and    -   b) instructions for use of the pharmaceutical composition to        treat a subject afflicted with a depressive or stress disorder.

The present invention further provides a pharmaceutical compositioncomprising an amount of a phosphatase inhibitor of the presentapplication for use in treating a subject afflicted with a depressive orstress disorder.

The present inventions also provides a therapeutic package fordispensing to, or for use in dispensing to, a subject afflicted with adepressive or stress disorder, which comprises:

-   -   a) one or more unit doses, each such unit dose comprising a        phosphatase inhibitor of the present application; and    -   b) a finished pharmaceutical container therefor, said container        containing said unit dose or unit doses, said container further        containing or comprising labeling directing the use of said        package in the treatment of said subject.

The analogs of LB-100 disclosed herein have analogous activity to LB-100and behave similarly in the assays disclosed herein.

The present invention provides a pharmaceutical composition comprising acompound of the present invention for use in treating a depressive orstress disorder.

In some embodiments, the pharmaceutical composition wherein thepharmaceutically acceptable carrier comprises a liposome.

In some embodiments, the pharmaceutical composition wherein the compoundis contained in a liposome or microsphere.

In some embodiments of any of the above methods or uses, the subject isa human. In some embodiments of any of the above methods or uses, thesubject is an adult patient, pediatric patient, male patient and/orfemale patient.

In some embodiments of any of the above methods or uses, the compound isorally administered to the subject.

In one embodiment, the subject afflicted with the depressive or stressdisorder has been affirmatively diagnosed to have the condition. Forexample, a patient afflicted with PTSD means a patient who has beenaffirmatively diagnosed to have PTSD. The diagnosis of the disorder canbe effected using any of the appropriate methods known in the art. Forexample, PTSD can be diagnosed by psychological evaluation and/or bymeeting the criteria in the Diagnostic and Statistical Manual of MentalDisorders (DSM-5), published by the American Psychiatric Association.

In an embodiment of the present invention the method includes the stepof identifying or determining whether the subject is afflicted with thedepressive or stress disorder.

The compounds used in the method of the present invention are proteinphosphatase 2A (PP2A) inhibitors. Methods of preparation may be found inLu et al., 2009a-b; U.S. Pat. No. 7,998,957 B2; and U.S. Pat. No.8,426,444 B2. Compound LB-100 is an inhibitor of PP2A in vitro in humancancer cells and in xenografts of human tumor cells in mice when givenparenterally in mice. LB-100 inhibits the growth of cancer cells inmouse model systems.

In one embodiment of any of the above methods, the method consistingessentially of administering the compound.

As used herein, a “symptom” associated with the depressive or stressdisorder includes any clinical or laboratory manifestation associatedwith depressive or stress disorders and is not limited to what thesubject can feel or observe.

As used herein, “treatment of the diseases” or “treating”, e.g. ofreperfusion injury, encompasses inducing prevention, inhibition,regression, or stasis of the disease or a symptom or conditionassociated with the disease.

As used herein, “inhibition” of disease progression or diseasecomplication in a subject means preventing or reducing the diseaseprogression and/or disease complication in the subject.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. Thus, C₁-C_(n) as in “C₁-C_(n) alkyl”is defined to include groups having 1, 2 . . . , n−1 or n carbons in alinear or branched arrangement, and specifically includes methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl andso on. An embodiment can be C₁-C₂₀ alkyl, C₂-C₂₀ alkyl, C₃-C₂₀ alkyl,C₄-C₂₀ alkyl and so on. An embodiment can be C₁-C₃₀ alkyl, C₂-C₃₀ alkyl,C₃-C₃₀ alkyl, C₄-C₃₀ alkyl and so on. “Alkoxy” represents an alkyl groupas described above attached through an oxygen bridge.

The term “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present. Thus, C₂-C_(n) alkenyl isdefined to include groups having 1, 2 . . . , n−1 or n carbons. Forexample, “C₂-C₆ alkenyl” means an alkenyl radical having 2, 3, 4, 5, or6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, forexample, 3 carbon-carbon double bonds in the case of a C₆ alkenyl,respectively. Alkenyl groups include ethenyl, propenyl, butenyl andcyclohexenyl. As described above with respect to alkyl, the straight,branched or cyclic portion of the alkenyl group may contain double bondsand may be substituted if a substituted alkenyl group is indicated. Anembodiment can be C₂-C₁₂ alkenyl, C₃-C₁₂ alkenyl, C₂-C₂₀ alkenyl, C₃-C₂₀alkenyl, C₂-C₃₀ alkenyl, or C₃-C₃₀ alkenyl.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present. Thus, C₂-C_(n) alkynyl is defined to include groups having1, 2 . . . , n−1 or n carbons. For example, “C₂-C₆ alkynyl” means analkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triplebond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triplebonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds.Alkynyl groups include ethynyl, propynyl and butynyl. As described abovewith respect to alkyl, the straight or branched portion of the alkynylgroup may contain triple bonds and may be substituted if a substitutedalkynyl group is indicated. An embodiment can be a C₂-C_(n) alkynyl. Anembodiment can be C₂-C₁₂ alkynyl or C₃-C₁₂ alkynyl, C₂-C₂₀ alkynyl,C₃-C₂₀ alkynyl, C₂-C₃₀ alkynyl, or C₃-C₃₀ alkynyl.

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 10 atoms in each ring, wherein at leastone ring is aromatic. Examples of such aryl elements include phenyl,naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthrylor acenaphthyl. In cases where the aryl substituent is bicyclic and onering is non-aromatic, it is understood that attachment is via thearomatic ring. The substituted aryls included in this invention includesubstitution at any suitable position with amines, substituted amines,alkylamines, hydroxys and alkylhydroxys, wherein the “alkyl” portion ofthe alkylamines and alkylhydroxys is a C₂-C_(n) alkyl as definedhereinabove. The substituted amines may be substituted with alkyl,alkenyl, alkynl, or aryl groups as hereinabove defined.

Each occurrence of alkyl, alkenyl, or alkynyl is branched or unbranched,unsubstituted or substituted.

The alkyl, alkenyl, alkynyl, and aryl substituents may be unsubstitutedor unsubstituted, unless specifically defined otherwise. For example, a(C₁-C₆) alkyl may be substituted with one or more substituents selectedfrom OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such asmorpholinyl, piperidinyl, and so on.

In the compounds of the present invention, alkyl, alkenyl, and alkynylgroups can be further substituted by replacing one or more hydrogenatoms by non-hydrogen groups described herein to the extent possible.These include, but are not limited to, halo, hydroxy, mercapto, amino,carboxy, cyano and carbamoyl.

The term “substituted” as used herein means that a given structure has asubstituent which can be an alkyl, alkenyl, or aryl group as definedabove. The term shall be deemed to include multiple degrees ofsubstitution by a named substitutent. Where multiple substituentmoieties are disclosed or claimed, the substituted compound can beindependently substituted by one or more of the disclosed or claimedsubstituent moieties, singly or plurally. By independently substituted,it is meant that the (two or more) substituents can be the same ordifferent.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, “administering” an agent may be performed using any ofthe various methods or delivery systems well known to those skilled inthe art. The administering can be performed, for example, orally,parenterally, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery, subcutaneously, intraadiposally, intraarticularly,intrathecally, into a cerebral ventricle, intraventicularly,intratumorally, into cerebral parenchyma or intraparenchchymally.

The following delivery systems, which employ a number of routinely usedpharmaceutical carriers, may be used but are only representative of themany possible systems envisioned for administering compositions inaccordance with the invention.

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprolactones and PLGA's).

Other injectable drug delivery systems include solutions, suspensions,gels. Oral delivery systems include tablets and capsules. These cancontain excipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Implantable systems include rods and discs, and can contain excipientssuch as PLGA and polycaprylactone.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, zanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid),anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

As used herein, “pharmaceutically acceptable carrier” refers to acarrier or excipient that is suitable for use with humans and/or animalswithout undue adverse side effects (such as toxicity, irritation, andallergic response) commensurate with a reasonable benefit/risk ratio. Itcan be a pharmaceutically acceptable solvent, suspending agent orvehicle, for delivering the instant compounds to the subject.

The compounds used in the method of the present invention may be in asalt form. As used herein, a “salt” is a salt of the instant compoundswhich has been modified by making acid or base salts of the compounds.In the case of compounds used to treat an infection or disease, the saltis pharmaceutically acceptable. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as phenols. The salts can be made using an organic orinorganic acid. Such acid salts are chlorides, bromides, sulfates,nitrates, phosphates, sulfonates, formates, tartrates, maleates,malates, citrates, benzoates, salicylates, ascorbates, and the like.Phenolate salts are the alkaline earth metal salts, sodium, potassium orlithium. The term “pharmaceutically acceptable salt” in this respect,refers to the relatively non-toxic, inorganic and organic acid or baseaddition salts of compounds of the present invention. These salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or by separately reacting a purifiedcompound of the invention in its free base or free acid form with asuitable organic or inorganic acid or base, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

The present invention includes esters or pharmaceutically acceptableesters of the compounds of the present method. The term “ester”includes, but is not limited to, a compound containing the R—CO—OR′group. The “R—CO—O” portion may be derived from the parent compound ofthe present invention. The “R′” portion includes, but is not limited to,alkyl, alkenyl, alkynyl, heteroalkyl, aryl, and carboxy alkyl groups.

The present invention includes pharmaceutically acceptable prodrugesters of the compounds of the present method. Pharmaceuticallyacceptable prodrug esters of the compounds of the present invention areester derivatives which are convertible by solvolysis or underphysiological conditions to the free carboxylic acids of the parentcompound. An example of a pro-drug is an alkly ester which is cleaved invivo to yield the compound of interest.

The compound, or salt, zwitterion, or ester thereof, is optionallyprovided in a pharmaceutically acceptable composition including theappropriate pharmaceutically acceptable carriers.

As used herein, an “amount” or “dose” of an agent measured in milligramsrefers to the milligrams of agent present in a drug product, regardlessof the form of the drug product.

The National Institutes of Health (NIH) provides a table of EquivalentSurface Area Dosage Conversion Factors below (Table 1) which providesconversion factors that account for surface area to weight ratiosbetween species.

TABLE 1 Equivalent Surface Area Dosage Conversion Factors To Mouse RatMonkey Dog Man 20 g 150 g 3 kg 8 kg 60 kg From Mouse 1 ½ ¼ ⅙ 1/12 Rat 21 ½ ¼ 1/7 Monkey 4 2 1 ⅗ ⅓ Dog 6 4 1⅔ 1 ½ Man 12 7 3 2 1

As used herein, the term “therapeutically effective amount” or“effective amount” refers to the quantity of a component that issufficient to yield a desired therapeutic response without undue adverseside effects (such as toxicity, irritation, or allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of this invention. The specific effective amount will vary withsuch factors as the particular condition being treated, the physicalcondition of the patient, the type of mammal being treated, the durationof the treatment, the nature of concurrent therapy (if any), and thespecific formulations employed and the structure of the compounds or itsderivatives.

Where a range is given in the specification it is understood that therange includes all integers and 0.1 units within that range, and anysub-range thereof. For example, a range of 77 to 90% is a disclosure of77, 78, 79, 80, and 81% etc.

As used herein, “about” with regard to a stated number encompasses arange of +one percent to −one percent of the stated value. By way ofexample, about 100 mg/kg therefore includes 99, 99.1, 99.2, 99.3, 99.4,99.5, 99.6, 99.7, 99.8, 99.9, 100, 100.1, 100.2, 100.3, 100.4, 100.5,100.6, 100.7, 100.8, 100.9 and 101 mg/kg. Accordingly, about 100 mg/kgincludes, in an embodiment, 100 mg/kg.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg/m²” is a disclosure of 0.2 mg/m², 0.3mg/m², 0.4 mg/m², 0.5 mg/m², 0.6 mg/m² etc. up to 5.0 mg/m².

All combinations of the various elements described herein are within thescope of the invention.

For the foregoing embodiments, each embodiment disclosed herein iscontemplated as being applicable to each of the other disclosedembodiments. Thus, all combinations of the various elements describedherein are within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

Material and Methods

Experimental Subjects and Inescapable Shock Procedure

C57Bl/6J mice wild-type males of 4-7 weeks were used in accordance withthe guidelines of the French Agriculture and Forestry Ministry forhandling animal. They were housed at groups of 4-6 per cage with waterand food ad libitum available.

The inescapable shock procedure was previously described in Stamatakisand Stuber 2012. Briefly, we placed mice into standard mouse behavioralchambers (Imetronics) equipped with a metal grid floor. We let them tohabituate at the new environment for 5 m. In a 20 m session animalsreceived either 19 or 0 unpredictable foot shocks (1 mA, 500 ms) with aninterstimulus interval of 30, 60 or 90 s. We anesthetized mice forpatch-clamp electrophysiology 1 h, 24 h, 7 d, 14 d or 30 d after thesession ended.

Electrophysiology

Animals were anesthetized with Ketamine/Xylazine (50 mg/10 mg Kg-1 i.p.;Sigma-Aldrich, France). The preparation of LHb-containing brain sliceswas done in bubbled ice-cold 95% O₂/5% CO₂-equilibrated solutioncontaining (in mM): cholineCl 110; glucose 25; NaHCO₃ 25; MgCl₂ 7;ascorbic acid 11.6; Na+-pyruvate 3.1; KCl 2.5; NaH2PO4 1.25; CaCl2 0.5.Sagittal slices (250 μm) were stored at room temperature in 95% O₂/5%CO₂-equilibrated artificial cerebrospinal fluid (ACSF) containing (inmM): NaCl 124; NaHCO₃ 26.2; glucose 11; KCl 2.5; CaCl2 2.5; MgCl2 1.3;NaH₂PO₄ 1. Recordings (flow rate of 2.5 ml/min) were made under anOlympus-BX51 microscope (Olympus, France) at 32° C. Currents wereamplified, filtered at 5 kHz and digitized at 20 kHz. Access resistancewas monitored by a step of −4 mV (0.1 Hz). Experiments were discarded ifthe access resistance increased more than 20%.

The internal solution to measure GABAB-GIRK currents and neuronalexcitability contained 140 mM potassium gluconate, 4 mM NaCl, 2 mMMgCl₂, 1.1 mM EGTA, 5 mM HEPES, 2 mM Na₂ATP, 5 mM sodium creatinephosphate, and 0.6 mM Na3GTP (pH 7.3), with KOH. When we measured thesynaptic inhibitory or excitatory release the internal solutioncontained (in mM): CsCl 130; NaCl 4; MgCl₂ 2; EGTA 1.1; HEPES 5; Na₂ATP2; Na+-creatine-phosphate 5; Na₃GTP 0.6, and spermine 0.1. The liquidjunction potential was −3 mV. For cell-attached recordings the internalsolution consisted in ACSF.

Cell-attached recordings in ACSF were performed in GΩ seal with a Vc=0and Hc=0. Whole-cell voltage clamp recordings were achieved to measureGABAB-GIR currents in presence of Bicuculine (10 μM), NBQX (20 μM) andAP5 (50 μM). For agonist-induced currents, changes in holding currentsin response to bath application of baclofen were measured (at −50 mVevery 5 s). GABAB-GIRK currents were confirmed by antagonism with either10 μM of CGP54626, a specific GABAB antagonist or 1 mM Ba², a selectiveinhibitor of inward rectifiers. For the GTPγS experiment, 100 μM ofGTPγS was added to the internal solution in place of Na₃GTP.

For the Okadaic acid (OA) and the CaMKII inhibitor KN93 experiments,respectively 100 nM of OA or 10 μM of KN93 were added in the internalsolution. For the PP2A inhibitor LB100 experiments, LB100 (0.1 μM) wasapplied in the bath. Miniature excitatory postsynaptic currents (mEPSCs)were recorded in voltage-clamp mode at −60 mV in presence of bicuculine(10 μM) and TTX (1 μM). Miniature inhibitory postsynaptic currents(mIPSCs) were recorded (−60 mV) in presence of NBQX (20 μM) and TTX (1μM). EPSCs and IPSCs were evoked through an ACSF filled monopolar glasselectrode placed 200 μm from the recording site in the stria medullaris.AMPA:NMDA ratios of evoked-EPSC were obtained by AMPA-EPSC −60mV/NMDA-EPSCs at +40 mV. Rectification Index (RI) was computed byAMPAEPSC-60/AMPA-EPSC+40. For the experiments in which high-frequencystimulation trains were used to determine presynaptic releaseprobability, QX314 (5 mM) was included in the internal solution toprevent the generation of sodium spikes. Current-clamp experiments, wereperformed by a series of current steps (negative to positive) injectedto induce action potentials (5-10 pA injection current per step, 800ms).

In Vivo LB100 Treatment

For LB treatment experiments, mice were injected with LB (1.5 mg/Kgi.p.) or saline 6-8 hours after the FsE. The effects of the treatment onthe LHb excitability and GABAB-GIRKs currents as well as the behaviouralassay were assessed at day 1 or 7. For both electrophysiology andbehaviour once treated the animal were housed in groups of 4-6 and letundisturbed until the day of the test.

LB100 Local Injections

Animals were anesthetized with Ketamine (90 mg/kg)/Xylazine (15 mg/kgi.p.)(Sigma-Aldrich, France) 6-8 hours after the shock protocol andbefore bilateral injection with LB100 (1 μM) in the LHb: A-P: −1.7; M-L:±0.45; D-V: −3.1. Retrobeads (Lumafluor, Nashville, US) were added forpost histological identification of the injection site. Control animalswere simply injected with PBS and retrobeads in a final volume of300-500 nl. Brain slices from mice injected were directly examined underfluorescent microscope. Only animals with correct injections site wereincluded in the analysis. qRT-PCR LHb punches (2 mm diameter, 2 mmthick) were taken from male C57BL/6J mice (4-7 weeks). Quantitativeanalysis of GIRK1, GIRK2, GIRK3 and GIRK4 mRNA levels was performed.

Quantitative Immunoelectron Microscopy

The subcellular distribution of Girk2 and GABABR1 was measured usingpreembedding immunoelectron microscopy and quantitative analysisapproaches, as previously described (Arora et al 2011).

Other Aversion Induced Protocols

Odor Predator Exposure

Mice were exposed to a predator odor by presenting for 5 min a cottonball soaked with red fox urine (5 ml Red fox P; Timk's, SafarilandHunting Corp., Trappe, Mass.) placed in a plastic container (holesequipped) in a corner of a transport cage. For the control group insteadfox urine we added 5 ml of water. One hour after the procedure, the micewere anesthetized for the vitro recordings.

Restraint Stress

A ventilated 50 ml falcon tube placed at the center of a transport cagewas employed to constrain the mice for 1 h (from 9.00 to 10.00 am).Control animals were let undisturbed in a transport cage for the sameamount of time. The mice were anesthetized for the vitro recordings, onehour after the procedure.

Behavioral Assays.

All behavioral tests were conducted during the light phase (8:00-19:00),1 or 7 days after the shock procedure.

Re-exposure to the Context Test

For this experiment mice were re-exposed 24 h after to the chamber wherethey received (or not) shocks for a total duration of 5 m. Onlineanalysis of the freezing was performed by viewing a video monitor in aroom separate from the mice during the test period. Offline analysis wasperformed by a second observer. Freezing was defined as the absence ofvisible movement except that required for respiration (fluctuation inthe volume of the thorax) (score: 1). Scanning was assessed when theanimal showed a sole movement of the head to scan the environment in adefensive position (score: 0.5). The behavior was scored according to a5-sec instantaneous time-sampling procedure every 25 sec (Fanselow andBolles 1979). The observer scored the animal as freezing, scanning oractive at that instant and then proceeded to the next chamber. Afterchamber 6, the observer started again with chamber 1. Thus each animalwas scored every 5-sec, yielding 10 observations for each mouse fortotal 5 minutes session. The cumulative score were converted intopercent time freezing by dividing the number of freezing-scanningobservations by the total number of observations for each mouse.

Forced Swim Test

Forced swim test was conducted under normal light condition aspreviously described (Meye, Valentinova, Lecca et al 2015). Mice wereplaced in a cylinder of water (temperature 23-25° C.; 14 cm in diameter,27 cm in height for mice) for six min. The depth of water was set toprevent animals from touching the bottom with their hind limbs. Animalbehavior was videotracked from the top (Viewpoint, France). The latencyto the first immobility event and the immobility time of each animalspent during the test was counted online by two independent observerblind of the animal treatments. Immobility was defined as floating orremaining motionless, which means absence of all movement except motionsrequired to maintain the head above the water.

Analysis and Drugs

All drugs were obtained from Abcam (Cambridge, UK), Tocris (Bristol, UK)and Sigma (Lyon, France) and dissolved in water, except for TTX (citricacid 1%). Analysis for the electrophysiology data was performed inblocks depending on the experiment. Online/offline analyses wereperformed using IGOR-6 (Wavemetrics, US) and Prism (Graphpad, US).Sample size required for the experiment was empirically tested byrunning pilots experiments in the laboratory. Data distribution waspreviously tested by the Kolgorov Smirnoff and D'Agostino Pearson.Depending by the distribution parametric or not parametric test wereused. Single data points are always plotted. Compiled data are expressedas mean±s.e.m. Significance was set at p<0.05 using Student's t-test,one or two-way ANOVA with multiple comparison when applicable. MannWithney test was used when required.

Example 1. A Single Foot Shock Exposure Produces Depressive-likeBehaviors and Increases LBb Neuronal Excitability

The LHb mediate different behavioral phenotypes mostly linked toaversive-related conditions (Lecca et al., 2014). Increased excitatoryoutput from the LHb is crucial for driving aversion to external contextand avoidance behaviors (Stamatakis and Stuber, 2012). However, whetheradaptations occurring in the LHb early after the exposure to an aversiveexperience are ultimately important for behavioral responses is unknown.

To test this, behavioral consequences upon the exposure to a singlesession of foot-shocks (FsE), a protocol that if stronger drives acutelearned helplessness, were examined (Hammack et al., 2012; Li et al.,2011). C57/BL6J mice were exposed to none or 19 unpredictablefoot-shocks in a single 20 minutes session (FIG. 1A)(Stamatakis andStuber, 2012). Animals were re-exposed to the context 24 hours after theprotocol, and as predicted, mice presented a high level of freezing, abehavioral trait typical of fear conditioning (FIG. 1B)(Luthi andLuscher, 2014). The same group of animals was then tested 7 days afterthe FsE by employing the forced swim test (FST) paradigm to assessdepressive-like states (Porsolt et al., 1977). FsE resulted indepressive-like behaviors, with a reduced latency to first immobilityand increased total immobility in the FST, indicating that a singleexposure to a session of aversive stimuli have important behavioralconsequences (FIG. 1C).

Hyperexcitability in the LHb contributes to disorders characterized bydepressive-like phenotypes including depression and addiction (Li etal., 2011; Shabel et al., 2014; Stamatakis and Stuber, 2012) (Meye,Valentinova, Lecca et al., 2015). Use of drugs or alcohol producesrewarding effects followed by aversive effects including depression. TheLHb has also been implicated in aversive effects caused by drug oralcohol addiction or withdrawal from drug or alcohol addiction (Jhou, T.C. et al. 2013; Meye, F. J. et al. 2015).

It was predicted that the establishment of depressive-like traits afterFsE relies, at least in part, to modifications in LHb function. To testthis, LHb neurons spontaneous action potentials were measured incell-attached mode 1 hour after FsE (FIG. 1D).

It was found that action potentials were more frequent in micepreviously exposed to footshock than in controls (FIG. 1E).Cell-attached recording, although non-invasive may result in alterationsin the firing rate (Alcami et al., 2012). To rule out potentialtechnical pitfalls, firing activity using whole-cell recording wascompared allowing for an alternative assessment of FsE on LHb neuronsactivity. It was found that that when plotting the number of actionpotentials in function of progressively larger current injection, LHbneurons in the FsE presented a larger number of action potential (FIG.1F).

Altogether this indicates that FsE produces depressive-like states inmice as well as hyperexcitability of LHb neurons.

Example 2. Exposure to Aversive Stimuli Depresses GABAB-GIRK Function inthe LHb

Multiple mechanisms may underlie neuronal excitability includingmodifications in the balance between excitation/inhibition as well asmodifications in ion conductance. To test the underlying cellularmechanisms for cell hyperexcitability in LHb upon FsE, miniatureexcitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) wereinitially recorded 1 hour after the FsE (FIG. 2A, B). It was found thatquantal excitatory and inhibitory synaptic transmission remainedunaffected in both frequency and amplitude (FIG. 2A, B). Additionally,no modifications were found in the postsynaptic strength of AMPA orNMDA-mediated transmission nor subunit composition as AMPA/NMDA ratiosand rectification index were comparable among experimental groups(Figure S1A). To determine potential FsE-induced modifications in theefficacy of presynaptic neurotransmitter release, evoked transmissionwas examined placing a stimulating electrode in the stria medullaris. Toprobe presynaptic function, evoked E/IPSCs were examined with trains athigh frequency (10 stimuli delivered at 20 Hz) (Zucker and Regehr,2002). It was found that at both excitatory and inhibitory trains ofsynaptic responses were comparable in slices between control and FsEmice (Figure S1B).

GIRK channels hyperpolarize neurons in response to activation of GABABreceptors, thereby providing a fine control of neuronal excitability(Luscher and Slesinger, 2010). Plasticity of GABAB-GIRK signaling iscritical for specific behavioral adaptations (Hearing et al., 2013;Padgett et al., 2012). However the functional relevance of GABAB-GIRKsignaling in LHb remains understudied. Bath application of a saturatingdose of the GABAB-R agonist baclofen (100 μM) evoked an outward current(IBaclofen) that showed little desensitization during continuous drugapplication (Figure S2A). The outward response was readily reversed bythe GABAB-R antagonist CGP54626 (10 μM; Figure S2A & E). Baclofenproduced outward responses in a dosedependent manner, with an EC50 of10.2 μM (Figure S2B). I-Baclofen correlated with a decrease in inputresistance (Figure S2C) consistently with activation of K+ conductance.Accordingly, I-Baclofen was abolished in presence of low barium (Ba2+)concentrations (1 mM) indicating that inwardly rectifying K+ channelsare likely the effectors of GABAB activation in the LHb (Figure S2D).The LHb of mice were microdissected, and whole tissue real-time PCR wasperformed. It was found that GIRK1-4 subunits were expressed in the LHb,supporting a functional GABAB-GIRK signaling in the LHb (Figure S2E).

Altogether these results indicate that GABAB-GIRK signaling provide afunctionally relevant inhibitory control of LHb neurons activity. Totest whether FsE alter the GABAB-GIRK component, whole-cell recordings 1hour later in LHb neurons were performed, and recorded I-Baclofen in thedifferent experimental groups. I-Baclofen in slices from FsE mice wassmaller compared to control mice (FIG. 2C). In order for cellularmodifications to be behaviorally relevant, they should not fade offrapidly, providing an important substrate for reorganization of complexneural circuits (Mameli et al., 2009; Chen et al., 2008). To determinethe longevity of the FsE-induced adaptations, it was examined whetherGABAB-GIRK signaling was reduced after 1, 7, 14, and 30 days followingFsE. FsEevoked reduction in I-Baclofen persisted up to 14 days after theparadigm, and it was instead comparable to control values one monthafter the session (FIG. 2C). The modifications induced after aversiveexperiences are specific for the GABAB-GIRK signaling. Indeed, theI-Baclofen evoked in the presence of Ba2+ was comparable between controland foot-shock-exposed mice (FIG. 2D). This indicates that solely theGABAB-GIRK, Ba2+-sensitive, component is altered by aversive experience.

Altogether these findings indicate that FsE exposure selectively andpersistently modifies GABAB-GIRK signaling in the LHb without alteringsynaptic transmission onto LHb neurons.

Example 3. GABAB-GIRK Plasticity in the LHb for Generalised NegativeExperience

Conditions of heightened stress can represent a risk factor for thedevelopment of neuropsychiatric disorders spanning to post-traumaticstress disorders to depression (Radley et al., 2011). Heightened stressparadigms, not driven by painful stimuli, but nevertheless capable totrigger avoidance and depressive-like behaviors, were tested todetermine if they modified GABAB-GIRK signaling in LHb (Buynitsky andMostofsky, 2009; Takahashi et al., 2005). The odor predator stressparadigm involves the use of a natural stimulus, as opposed tofoot-shock, to provide a condition with heightened stress. Mice weresubmitted to fox urine for five minutes and slices containing the LHbwere prepared 1 hour after exposure. Similarly to FsE mice, I-Baclofenwas depressed in animals exposed to the odor predator (FIG. 3A).Furthermore, restraint stress, also used to model depressive-likebehaviors in rodents (Hammack et al., 2012), similarly reducedI-Baclofen in LHb neurons (FIG. 3B).

Taken together these findings indicate that the reduction in GABAB-GIRKsignaling may represent a common cellular substrate for the encoding ofaversive stimuli.

Example 4. FsE-induced GABAB-GIRK Redistribution in the LHb

Mechanistically, the reduction of GABAB-GIRK function upon aversiveexperience may occur via alternative mechanisms including modificationsin G protein coupling (Labouebe et al., 2007), scaffolding proteinadaptations (Tiao et al., 2008) or internalization of the receptors orreceptor/effector complex (Hearing et al., 2013; Padgett et al., 2012).To test the latter scenario, quantitative immunoelectron microscopy wasemployed to compare the subcellular distribution of GABAB1 and GIRK2 inthe LHb in control and FsE mice (FIG. 4A, B). It was found that noglobal changes in the total GABAB1 and GIRK2 immunolabeling betweendifferent experimental groups occurred one hour after FsE (FIG. 4C, D).Membrane GABAB1 and GIRK2 were mainly located at extrasynaptic sites(FIG. 4B). FsE led to a significant reduction in membrane immunolabelingof both GABAB1 and GIRKs, with a corresponding increase in theirintracellular labeling (FIG. 4C, D). These finding supports a scenarioin which GABAB-Rs and GIRKs traffic out the plasma membrane as amacrocomplex (Boyer et al., 2009; Padgett et al., 2012).

To further assess a reduction in the functional expression of GIRKchannels, the effect of intracellular GTPγS was examined. GTPγS is anon-hydrolysable form of guanosine triphosphate (GTP) that allowsbypassing GPCR activation and constitutively activate GIRK channels viaGb/g-dependent mechanisms (Logothetis et al., 1987). In light of ourimmunogold labeling, it was predicted that the intracellular dialysis ofGTPγS would elicit smaller outward currents in LHb neurons from FsE micecompared to animals of the control group. In control neurons, intrapatchdialysis of GTPγS (100 μM) led to slow activation of a large outward K+current sensitive to extracellular Ba², indicative of GIRK channelsactivation (FIG. 4E). LHb neurons from slices of FsE mice showed reducedGTPγS-induced Ba2+-sensitive currents (FIG. 4E). These results suggestthat, in LHb neurons, FsE diminishes the functional membrane expressionof GIRK channels coupled to GABAB receptors.

Example 5. PP2A Activity in LHb Gates FsE-Evoked GABAB-GIRKInternalisation

The phosphorylation of specific serine residues on the B1 and B2subunits of the GABAB receptors is determinant to control GABAB-GIRKcomplex function (Guetg et al., 2010; Terunuma et al., 2010).Specifically, the dephosphorylation of serine 783 (S783) in the GABAB2is associated with pharmacological and drug-driven reduction in GABABreceptors surface expression in neurons (Padgett et al., 2012; Terunumaet al., 2010). This process relies on the increased activity of proteinphosphatase 2A, which constitutively maintains low GABAB-GIRK signaling(PP2A; (Hearing et al., 2013; Padgett et al., 2012; Terunuma et al.,2010)). Hence, FsE may enhance PP2A activity in LHb neurons, promotingthe sustained intracellular localization of GABABGIRK complexes. To testthis, the effect of acute intracellular inhibition of PP1/PP2Aphosphatases was examined by postsynaptic dialysis of okadaic acidthrough the patch pipette (OA; 100 nM). In control mice and in presenceof OA, I-Baclofen was comparable to naïve condition indicating thatPP1/PP2A activity does not provide substantial control of GABABR-GIRKssignaling at baseline (FIG. 5A). In contrast, in FsE mice intracellulardialysis of OA led to I-Baclofen amplitudes comparable to control mice(FIG. 5A). This suggests that in the LHb, OA dialysis, and thereforePP1/PP2A inhibition, promptly restore GABAB-GIRK signaling after FsE.

An alternative pathway controlling GABAB surface is the CaMKII-mediatedphosphorylation at S867 of GABAB1 (Guetg et al., 2010). To assess apotential involvement of CaMKII in FsE-evoked GABAB-GIRK reduction, theeffect of the CaMKII inhibitor, KN93 (10 μM), was examined. Similarly toOA, the intracellular dialysis of KN93 did not affect I-Baclofen atbaseline. In contrast, KN93 failed to recover I-Baclofen in slices fromFsE mice, as currents were significantly smaller in the FsE group (FIG.5B). These results suggest a crucial role of PP1/PP2A activity for aconstitutive maintenance of internalized GABABRs and GIRKs. The use ofOA to inhibit phosphatases activity is however limited to itsintracellular use, as it lacks membrane permeability properties. Recentadvances in drug development and pharmacotherapy has targeted PP2Aactivity. Newly developed molecules have been generated to specificallyinhibit PP2A activity with the goal of enhancing effectiveness of cancertreatments that damage DNA or disrupt components of cell replication (Luet al., 2009b). This body of work has led to generate effective membranepermeable PP2A inhibitors, such as LB-100, that are now clinicallyrelevant for cancer therapy.

Specific inhibition of PP2A activity in acute brain slices by LB-100(0.1 μM) was tested. When in presence of LB-100 in the bath, I-Baclofenin control and FsE mouse slices were comparable (FIG. 5C). These datastrengthen the hypothesis that persistent PP2A activity after FsEmediates GABAB-GIRK signaling reduction. These results show that FsEleads to GABABRs and GIRK channels internalization and functionalreduction.

To address whether inhibition of PP2A activity rescues solely GABAB-Rsexpression and function, or also GIRKs expression and function,intrapatch dialysis of GTPγS were employed to again activate GIRKsindependently of GABABRs. It was found that, in presence of LB-100,I-Baclofen in LHb neurons from FsE mice were comparable to the controlgroup (FIG. 5D). Altogether, this indicates that, not only GABAB-Rs, butalso GIRK signaling or functional G protein coupling is restored wheninhibiting PP2A activity (FIG. 5C, D).

Taken together, these findings suggest that foot shock experiencetriggers a phosphatase-dependent downregulation of GABAB-GIRK complexfrom the plasma membrane of LHb neurons, thereby reducing GABABR-GIRKsignaling.

Example 6. Loss of GADAbR-dependent Inhibition of LHb Neuron Firing

GABAB-mediated activation of Kir3 channels produces slow inhibitorypostsynaptic potentials by inducing K+ efflux thereby hyperpolarizingthe cell membrane and shunting neuronal firing (Pinard et al., 2010).The data herein suggests that FsE increases the activity of LHb neurons,and provide a reduction in GABAB-GIRK signaling (FIGS. 1E & F; 2C).Additional experiments were performed to determine if the two functionalmodifications were causally linked.

To investigate the functional consequences of depressed GABAB-GIRKcurrents in LHb neurons of FsE, baclofen-evoked modifications in LHbneurons output firing by recording neurons were examined in thecell-attached mode (FIG. 6A). It was predicted that a reduction inGABAB-GIRK signaling would not only increase firing of LHb neurons atbaseline (FIG. 1E), but also weaken GABAB-GIRK-induced firingsuppression. Bath application of baclofen in slices from controlanimals, produced an almost-complete reduction of LHb neuronal firing,which was promptly recovered and overshooted by CGP52536 (FIG. 6A).Conversely, in slices from FsE mice, LHb neurons presented higher basalfiring in average (see also FIG. 1E), which was only mildly reduced bybaclofen bath application (FIG. 6A).

To further stress these finding, a set of experiments were performed inwhich whole-cell mode and current steps (20 pA) were injected to elicita train of action potentials in LHb neurons over time. As expected,baclofen bath applications significantly depressed firing of LHb neuronsrecorded from control mice (FIG. 6B). By contrast, the same saturatingdose of baclofen only partially decreased firing of LHb neurons in FsEanimals (FIG. 6B). In order to establish whether rescuing GABAB-GIRKsignaling causally recovers FsE-evoked hyperexcitability, we examinedthe effect of PP2A inhibition on cell excitability in control and FsEmice. As previously shown (FIG. 1F), FsE increased the input-output(I-O) relationship, and LHb neurons after FsE present a top/left-shiftedI-O curve (FIG. 6C). In contrast, when LB-100 was applied, allowinginhibition of PP2A activity, I-O curves in slices from FsE mice werecomparable to control conditions (FIG. 6C).

Altogether these results demonstrate that a loss of GABABR-GIRK currentsin LHb neurons renders neurons more excitable and less sensitive tobaclofen-driven inhibition. Moreover, PP2A inhibition may represent aviable pharmacological tool to rescue FsE-evoked cellular modificationsin the LHb.

Example 7. PP2A Inhibition In Vivo Restores GAB-GIRK Function andAmeliorates FsEdriven Depressive Phenotypes

It has been shown herein that FsE decreases GABAB-GIRK signaling andpromotes hyperexcitability of LHb neurons, through a PP2A-dependentinternalization of the GABAB-GIRK complex. As a consequence, it waspredicted that inhibition of PP2A activity in vivo: i. would normalizeFsE-induced GABAB-GIRK reduction and hyperexcitability and ii. wouldrescue FsE-driven depressive-like phenotype as a consequence of therecovered LHb activity. To test the first prediction, FsE mice weresystemically treated them with vehicle or LB-100 (1.5 mg/Kg) 6-8 hoursafter the paradigm, a time point at which I-Baclofen is reduced (FIG.2A-C). At this concentration LB-100 efficiently inhibits PP2A activityfor about 12 hours, with a peak of its activity at ˜8 hours (Lu et al.,2009b). LHb-containing slices were prepared one or seven days after FsE.It was observed that FsE vehicle-treated mice had significantly smallerI-Baclofen compared to control vehicle-treated animals. In contrast,I-Baclofen was comparable between the control and FsE group aftersystemic injections of LB-100 at one day as well as seven days after FsE(FIG. 7C, D). This indicates that, in vivo inhibition of PP2A activityrescues the FsE-evoked plasticity of GABAB-GIRK signaling.

It was next explored whether the rescue of GABAB-GIRK currents by PP2Ainhibition was also accompanied by a functional rescue of LHb neuronalactivity. LHb neuronal excitability were recorded in whole-cell mode.FsE animals vehicle-treated, as previously shown (FIG. 1F; 6C),exhibited a top/left-shift in the I-O curve at one day and seven dayspost FsE (FIG. 7E). Conversely, in FsE mice, LB-100 systemic injectionnormalized LHb neuronal excitability (FIG. 7F), highlighting a crucialrole of persistent PP2A activity in controlling GABAB-GIRKs membranelevels, that is turn fundamental for neuronal activity.

If PP2A-dependent FsE-evoked GABAB-GIRK plasticity and hyperexcitabilityis a necessary for behavioral responses after aversive experience, PP2Ainhibition may represents a viable intervention to rescue FsE-drivendepressive-like phenotype. To this end, mice were subjected (or not) toFsE and it was found that one day after re-exposure to the contextproduced high levels of freezing only in FsE mice (FIG. 7F). Thisbehavior was not altered by systemic PP2A inhibition (FIG. 7F). Sevendays after FsE, mice were tested in the FST paradigm to assess theirdepressive-like phenotype. In FsE vehicle-treated mice, we found reducedlatency to the first immobility along with an increased totalimmobility. In contrast the treatment with LB-100 led to a behavioralnormalization of FsE-driven depressive-like phenotype, as latency to thefirst immobility and total immobility were comparable between controland FsE group (FIG. 7G). These findings establish a link between theGABAB-GIRK function, their role in controlling LHb neuronactivity andthe behavioral response to the exposure to an aversive condition.

Example 8. Administration of LB-100

An amount of compound LB-100 is administered to a subject afflicted witha depressive disorder, stress disorder or addiction. The amount of thecompound is effective to treat the subject.

An amount of compound LB-100 is administered to a subject following atraumatic event experienced by the subject. The amount of the compoundis effective to prevent a depressive or stress disorder from afflictingthe subject following the traumatic event or reduce the severity of adepressive or stress disorder that afflicts the subject following thetraumatic event.

An amount of compound LB-100 is administered to a subject afflicted witha depression or PTSD. The amount of the compound is effective to treatthe subject.

Example 9. Administration of LB-100 Analogs

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject afflicted with adepressive disorder, stress disorder or addiction. The amount of thecompound is effective to treat the subject.

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject following atraumatic event experienced by the subject. The amount of the compoundis effective to prevent a depressive or stress disorder from afflictingthe subject following the traumatic event or reduce the severity of adepressive or stress disorder that afflicts the subject following thetraumatic event.

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject afflicted with adepression or PTSD. The amount of the compound is effective to treat thesubject.

Example 10. Administration to Substance Addicted Subjects

An amount of compound LB-100 is administered to a subject suffering froma depressive or stress disorder caused by an addictive substance orcaused by discontinuing use of an addictive substance. The amount of thecompound is effective to treat the subject.

An amount of compound LB-100 is administered to a subject suffering froma depressive or stress disorder induced by withdrawal from the use of anaddictive substance. The amount of the compound is effective to treatthe subject.

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject suffering from adepressive or stress disorder caused by an addictive substance or causedby discontinuing use of an addictive substance. The amount of thecompound is effective to treat the subject.

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject suffering from adepressive or stress disorder induced by withdrawal from the use of anaddictive substance. The amount of the compound is effective to treatthe subject.

An amount of compound LB-100 is administered to a subject suffering froma depressive or stress disorder and addicted to an addictive substance.The amount of the compound is effective to treat the subject.

An amount of any one of the compounds of the present invention, whichare analogs of LB-100, is administered to a subject suffering from adepressive or stress disorder and addicted to an addictive substance.The amount of the compound is effective to treat the subject.

Discussion

Cellular Mechanisms Underlying GABAB-GIRK Plasticity in the LHb

Pharmacological approaches revealed that most (˜80%) of the directinhibitory effect of GABABR activation in LHb neurons can be attributedto GIRK channel activation. Importantly, exposure to aversive eventsselectively depressed the dominant GIRKdependent component ofI-Baclofen, without modifying the GABAB-dependent GIRK-independentpathway. The FsE-induced weakening of GABAB-GIRK currents in LHb neuronscould rely on alternative processes including modifications in G proteincoupling (Labouebe et al., 2007) or internalization of the receptors orchannels (Guetg et al., 2010; Hearing et al., 2013; Padgett et al.,2012; Terunuma et al., 2010). In support of the latter scenario,quantitative immunogold electron microscopy revealed a significantreduction in the membrane expression of GABAB receptors and GIRKchannels in LHb neurons previously exposed to a foot-shock paradigm. Theendocytosis rate of GABAB-R₅ in cortical, hippocampal, and GABA neuronsof the midbrain, requires the balance of AMP-activated protein kinase(AMPK)-dependent phosphorylation of GABAB2-S783 and PP2A-dependentdephosphorylation of GABAB receptors (Gonzalez-Maeso et al., 2003;Padgett et al., 2012; Terunuma et al., 2010). The FsE evoked persistentdepression of the GABAB-GIRK signaling and the rapid recovery obtainedwith phosphatase inhibitors suggests that PP2A activity controls thebalance between membrane and intracellular GABAB receptors (Padgett etal., 2012).

Although the data suggests that CaMKII is not involved in maintainingGABAB-GIRK internalization, one cannot rule out potential implicationsof CaMKII or PKA at different time points as they also target specificresidues of GABAB-R₅ subunits and control their membrane expression andsurface stability (Gonzalez-Maeso et al., 2003; Guetg et al., 2010;Terunuma et al., 2010). While these mechanisms control traffic of GABABreceptors, it was found that endocytosis of GIRK channels also occursafter aversive experience. Recent evidence suggests that GIRK channelsinternalize via association with GABAB receptors in a macromolecularsignaling complex (Hearing et al., 2013; Padgett et al., 2012). Indeed,both in the ventral tegmental area and medial prefrontal cortex drugexperience promotes the internalization of GABAB-Rs and GIRK channels(Lalive et al., 2014; Lavine et al., 2002; Nobles et al., 2005; Riven etal., 2006) and can traffic together through intracellular compartments(Clancy et al., 2007).

G-protein-insensitive inwardly rectifying K+ channels (kir2) bearsimilar PDZ domains and have been shown to be regulated by proteins ofthe postsynaptic density like SAP-97 and PSD-93, leading to increasedstability at the membrane (Leonoudakis et al., 2004; Leyland and Dart,2004). It has been shown that the LHb contains protein of the four GIRKssubunits GIRK1-4. As shown previously, GIRK2c and GIRK3 contain aC-terminal tail containing a PDZ binding domain implicated in GIRKchannel trafficking (Lunn et al., 2007). Despite high PDZ structuresimilarity, GIRK2c and GIRK3 do not interact with SAP-97 or PSD-95(Hibino et al., 2000; Nehring et al., 2000). However, an interaction hasbeen revealed with sorting nexin protein 27 (SNX27; (Lunn et al.,2007)), which specifically recognizes GIRK2c and GIRK3, but not Kir2.1subunits (Balana et al., 2011). SNX27 has been reported to mediate bothendosome-directed trafficking of GIRK channels and membrane traffickingof receptors like β2-adrenoreceptor (Lauffer et al., 2010), andtherefore represents a potential candidate in mediating GIRK channelplasticity. Whether alternative processes such as phosphorylation ofGIRK subunits changing their intrinsic properties affecting theirefficacy independently of the receptor-effector coupling remains to beestablished (Mao et al., 2004).

GABAB-GIRK Signaling Reduction and Functional Remodeling of NeuralCircuit

How does depression of GABAB-GIRK signaling in the LHb contribute to theLHb contact DA neurons in the ventral tegmental area, and, to a largeextent, midbrain GABA neurons (including RMTg and VTA)? It was alsofound that FsE-evoked GABAB-GIRK plasticity occurs throughout the LHb,with no apparent territorial specificity. This indicates that diverseneuronal populations in the LHb undergo similar changes that impact onparallel downstream midbrain targets, and be instrumental fordepressive-like phenotype (Meye Valentinova Lecca et al., 2015).

Behavioral Consequences after Aversive Experience?

GABAB-GIRK complexes control the excitability of many neuronalpopulations throughout the central nervous system in both physiologicaland pathological conditions (Luscher and Slesinger, 2010). We show that,in the LHb, pharmacological GABAB activation readily suppresses LHbfiring (FIG. 5A, B). This inhibitory control is lacking after aversiveexperience, thereby increasing the firing rate of LHb neurons. This mayhave profound impact on downstream targets as the endogenous activity ofLHb glutamatergic outputs conveys information related to aversion.Similarly to this study, exposure to a single session of unpredictedfoot-shock increase excitatory drive, likely via a presynaptic mechanismonto midbrain GABA neurons (Stamatakis and Stuber, 2012). Further,activation of LHb terminals onto midbrain midbrain neurons also driveconditioned place aversion (Lammel et al., 2012). This is in line withanatomical evidence that axons from the Indeed, Altered midbraininhibitory control has been implicated in several psychiatric disorders,including responses to aversive stimuli, depression, and addiction (Jhouet al., 2009; Luscher and Slesinger, 2010; Tye et al., 2013). Therefore,targeting inhibitory signaling may represent an innovative strategy fortherapeutic interventions in modern neuropsychiatry.

Molecular Mechanisms in LHb for FsE-induced Depressive-like Behaviors

The persistence of cellular and synaptic adaptations occurring earlyafter experience in specific brain structures remodels neural circuitproviding a substrate for behavioral adaptations (Belin and Everitt,2008; Mameli et al., 2009). The exposure to stressors can producelasting behavioral changes that resemble symptoms of depression in bothrodents and humans (Anisman and Zacharko, 1990; Rittenhouse et al.,2002).

Accordingly, posttraumatic stress disorder and major depressive disorderare highly comorbid psychopathologies (Bleich et al., 1997). We providedata supporting that FsE-driven GABAB-GIRK functional reduction occursrapidly and persist for up to two weeks. Furthermore, depressive-likesymptoms promoted by a single aversive experience can be rescued byblocking the PP2A activity, which is alone sufficient to normalizeGABAB-GIRK function in the LHb. This suggests a causal link betweenFsE-evoked cellular adaptations occurring in the LHb and the etiology ofdepressionlike phenotypes.

In line with these observations, dysfunction of LHb has been reported tobe critical in the etiology of mood disorders. Notably, in the learnedhelplessness rodent model of depression major synaptic modificationshave been suggested to mediate the maintenance of depressive-likestates. Specifically, increased excitatory drive onto LHb neurons, andan overexpression of the βCaMKII are both sufficient to promote neuronalhyperexcitability and depressive-like phenotype (Li et al., 2011; Li etal., 2013; Proulx et al., 2014). A hypothetical scenario is that theearly GABAB-GIRK reduction in the LHb due to a negative experience mayrepresent a gating mechanism for consequent adaptations suchβCaMKII-mediated synaptic modifications. Whether GABAB-GIRK plasticityrepresent a cellular substrate determinant for neuropsychiatricdisorders other than depression characterized by heightened LHb neuronalexcitability including drug abuse (Meye, Valentinova Lecca et al., 2015)remains to be tested.

The present findings indicate that aversion (or stress)-induceddepression of GABABR signaling in LHb neurons removes a cellular “brake”on LHb neuron activity that may provide a maladaptive excitation ofespecially GABA neurons of the midbrain, thereby mediating an inhibitionof DA neurons (Stamatakis and Stuber, 2012). Although more experimentsare necessary to understand the timeline of cellular events in the LHbduring highly stressful states, the rescue of GABA-GIRK signaling bytargeting for instance the PP2A might open potential strategies fortreating post-traumatic stress disorders or depressive-like symptoms.

Here, it was identified that GABA-GIRK signaling is a critical molecularlink between FsE and its long-lasting behavioral adaptations. FsEproduces delayed depressive-like behaviors and LHb neuronalhyperexcitability. We described that already after ˜1 hr following FsEor similar aversive experiences, GABAB-GIRK-mediated responses in LHbneurons are persistently reduced. FsE-induced diminished GABAB-GIRKfunction requires GABAB and GIRK trafficking to the endoplasm, andpersistent activation of the phosphatase PP2A. As a consequence, LHbneuron firing increased, and its modulation by baclofen was stronglyreduced. Reducing PP2A function in vitro as well as in vivo rescuesGABAB-GIRK signaling, and hyperexcitability. Finally, thepharmacological inhibition of PP2A rescues the FsE-drivendepressive-like phenotypes. These findings unravel early molecularmechanism in the LHb after negative experience and highlights potentialpharmacological targets to rescue behavioral adaptations driven byaversive events.

SUMMARY

Aversive conditions increase the excitability of LHb neurons, afundamental process for the development of depressive-like phenotypes indiverse neuropsychiatric disorders (Li et al., 2013) (Meye, Valentinova,Lecca 2015). Exposure to a single session of unpredicted aversivestimuli is sufficient to significantly weaken GABAB-GIRK signaling andto increase LHb neuronal activity. Taken at face value, thesemodifications alone are not sufficient to cause depression but rathermay represent an initial permissive step for widespread adaptations inthe LHb and downstream circuits contributing to the etiology of mooddisorders. The study of the early effects after a single exposure toaversive stimuli allowed us to dissect the precise molecular eventsunderlying the aversion-evoked plasticity of GABAB-GIRK signaling in theLHb. It was demonstrated that FsE-induced reduction of I-Baclofen arisesfrom an internalization of GABAB-Rs and GIRK channels that functionallydetermines hyperexcitability of LHb neurons.

Mechanistically these adaptations require a constitutive increase inPP2A activity, suggesting for a dephosphorylation-based internalizationof both GABAB and GIRKs. PP2A inhibition, such as by a PP2A inhibitor ofthe present application, restores GABAB-GIRK signaling, LHb neuronalexcitability and rescues FsE-driven depressive-like phenotype,indicating novel pharmacological targets to possibly contrast negativebehavioral states after a traumatic event.

Compound LB100 (see U.S. Pat. No. 7,998,957 B2) has anti-cancer activitywhen used alone (Lu et al. 2009a) and significantly potentiates in vivo,without observable increase in toxicity, the anti-tumor activity ofstandard cytotoxic anti-cancer drugs including temozolomide (Lu et al.2009b, Martiniova et al. 2010), doxorubicin (Zhang et al. 2010), anddocetaxel. LB100 was recently approved for Phase I clinical evaluationalone and in combination with docetaxel and is in clinical trial.However, the methods described herein employ LB100 for use in treatingor preventing depressive or stress disorders.

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What is claimed is:
 1. A method of treating major depression in asubject afflicted therewith comprising administering to the subject aneffective amount of a compound having the structure:

wherein bond α is present or absent; R₁ and R₂ together are ═O; R₃ isOH, O⁻, OR₉, O (CH₂)₁₋₆R₉, SH, S⁻, or SR₉, wherein R₉ is H, alkyl,alkenyl, alkynyl or aryl; R₄ is

where X is O, S, NR₁₀, N⁺HR₁₀or N^(+R) ₁₀R₁₀, where each R₁₀ isindependently H, alkyl, alkenyl, alkynyl, aryl,

 —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁, wherein each R₁₁ is independently H,alkyl, alkenyl or alkynyl; R₅ and R₆ taken together are ═O; R₇ and R₈are each H, or a salt, zwitterion, or ester thereof, so as to therebytreat the depressive or stress disorder in the subject.
 2. The method ofclaim 1, wherein the amount of the compound is effective to reduce aclinical symptom of the major depression in the subject.
 3. The methodof claim 1, wherein the treating comprises reducing the activity of thelateral habenula of the subject.
 4. The method of claim 1, wherein thetreating comprises reducing the activity of neurons in the lateralhabenula of the subject.
 5. The method of claim 1, wherein the treatingcomprises reducing neuronal hyperexcitability in the lateral habenula ofthe subject, or restoring normal GABAB-GIRK function in the lateralhabenula of the subject.
 6. The method of claim 1, wherein the treatingcomprises inhibiting phosphatase activity in the lateral habenula of thesubject.
 7. The method of claim 1, wherein the compound has thestructure

wherein bond αis present or absent; R₉ is present or absent and whenpresent is H, alkyl, alkenyl, alkynyl or phenyl; and X is O, NR₁₀,NH⁺R₁₀ or N⁺R₁₀R₁₀, where each R¹⁰ is independently H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl,

 —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂, where R₁₂ is H or alkyl, or a salt,zwitterion or ester thereof.
 8. The method of claim 1, wherein thecompound has the structure

or a salt or ester thereof.
 9. The method of claim 1, wherein the amountof the compound administered to the subject is 0.1 mg/m² to 5 mg/m². 10.The method of claim 9, wherein the amount of the compound isadministered once daily, once weekly, or once monthly.
 11. A method oftreating post-traumatic stress disorder (PTSD) in a subject afflictedtherewith in a subject following a traumatic event comprisingadministering to the subject an effective amount of a compound havingthe structure:

wherein bond α is present or absent; R₁ and R₂ together are ═O; R₃ isOH, O⁻, OR₉, O (CH₂)₁₋₆R₉, SH, S⁻, or SR₉, wherein R₉ is H, alkyl,alkenyl, alkynyl or aryl; R₄ is

where X is O, S, NR₁₀, N ⁺HR₁₀ or N³⁰ R₁₀R₁₀, where eachR_(10 is independently H, alkyl, alkenyl, alkynyl, aryl,)

 —CH₂CN, —CH₂CO₂R₁₁, or —CH₂COR₁₁, wherein each R₁₁ is independently H,alkyl, alkenyl or alkynyl; R₅ and R₆ taken together are ═O; R₇ and R₈are each H, or a salt, zwitterion, or ester thereof, so as to therebytreat the PTSD in the subject following a traumatic event.
 12. Themethod of claim 11, wherein the amount of the compound is effective toreduce a clinical symptom of the PTSD in the subject.
 13. The method ofclaim 11, wherein the treating comprises reducing the activity of thelateral habenula of the subject.
 14. The method of claim 11, wherein thetreating comprises reducing the activity of neurons in the lateralhabenula of the subject.
 15. The method of claim 11, wherein thetreating comprises reducing neuronal hyperexcitability in the lateralhabenula of the subject, or restoring normal GABAB-GIRK function in thelateral habenula of the subject.
 16. The method of claim 11, wherein thetreating comprises inhibiting phosphatase activity in the lateralhabenula of the subject.
 17. The method of claim 11, wherein thecompound has the structure

wherein bond α is present or absent; R₉ is present or absent and whenpresent is H, alkyl, alkenyl, alkynyl or phenyl; and X is O, NR_(10, NH)⁺R₁₀ or N⁺R₁₀R₁₀, where each R¹⁰ is independently H, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,

 —CH₂CN, —CH₂CO₂R₁₂, or —CH₂COR₁₂, where R₁₂ is H or alkyl, or a salt,zwitterion or ester thereof.
 18. The method of claim 11, wherein thecompound has the structure

or a salt or ester thereof.
 19. The method of claim 11, wherein theamount of the compound administered to the subject is 0.1 mg/m² to 5mg/m².
 20. The method of claim 19, wherein the amount of the compound isadministered once daily, once weekly, or once monthly.